Silencing of human c-myc oncogene expression by poly-DNP-RNA

Deregulation of c-myc oncogene expression drives the progression of many different types of cancer. Recent experimental data suggest that even brief inhibition of c-myc expression may be sufficient to permanently stop tumor growth and induce regression of tumors. Previous efforts in developing an inhibitor to silence the c-myc gene were hampered by low efficacy and lack of sequence specificity. Here, we report the synthesis of an antisense RNA inhibitor based on a new 21-nt sequence on a poly- DNP-RNA platform that can specifically inhibit cancer cell growth by silencing c-myc gene expression. Both c-myc mRNA and protein levels were significantly decreased in MCF-7 cells following treatment with this antisense DNP-RNA inhibitor. The control compounds with sense or mismatched sequence were inactive. When c-myc transgenic mice were each treated with a single dose of the antisense RNA inhibitor, in vivo silencing of c-myc gene expression was observed for up to 72 hours by real-time RT-PCR. Similar treatment of c-myc transgenic mice with unmodified (native) homologous small interfering RNA (siRNA) had no effect on the mRNA concentration of the c-myc gene. Injection of this short antisense poly-DNP-RNA into mice did not induce the synthesis of DNP-binding immunoglobulins in the host. The observed in vivo gene silencing by this antisense RNA inhibitor suggests its possible use as a therapeutic agent for cancers involving the deregulation of c-myc gene expression.     

The effect of dichloroacetic acid and trichloroacetic acid on DNA methylation and cell proliferation in B6C3F1 mice

The chlorine disinfection by-products, dichloroacetic acid (DCA) and trichloroacetic acid (TCA), are carcinogenic in mouse liver. We have previously reported that DCA and TCA induced DNA hypomethylation in mouse liver. In the present study, we determined the temporal association for DNA hypomethylation and cell proliferation. Female B6C3F1 mice were administered daily doses of 500 mg/kg DCA or TCA by gavage and sacrificed at 24, 36, 48, 72, and 96 hours after the first dose. The proliferating cell nuclear antigen-labeling index in the liver was increased at 72 and 96 hours by both DCA and TCA, that is, at 72 hours the index was 1.00 +/- 0.21, 0.51 +/- 0.11, and 0.095 +/- 0.016 for DCA, TCA, and the vehicle control, respectively. The mitotic index was also significantly increased at 96 hours. The promoter region for the c-myc gene was hypomethylated only at 72 and 96 hours and not at the earlier sacrifices. Similarly, the methylation of the c-myc gene in the kidney and urinary bladder was decreased only at 72 and 96 hours. In summary, enhancement of cell proliferation and decreased methylation of the c-myc gene were first observed simultaneously at 72 hours after the start of exposure. Thus, the results support the hypothesis that DCA and TCA induce DNA hypomethylation by inducing DNA replication and preventing the methylation of the newly synthesized strands of DNA.     

Preparation of trimers and tetramers of mixed sequence oligodeoxynucleoside methylphosphonates and assignment of configurations at the chiral phosphorus.

Synthesis of stereoregular DNA methylphosphonates has been accomplished for homo-oligomers, but remains a formidable problem for oligomers of a defined antisense target sequence. In this work, four trimer and tetramer deoxynucleoside methylphosphonates of mixed sequence (dACA, dCCAA, dAGGG, and dGCAT) were prepared by block coupling of diastereomerically pure dimers with either monomers or other diastereomerically pure dimers. These oligomers were separated chromatographically into individual diastereomers, and the configurations of the chiral methylphosphonate linkages were assigned. Three types of methods were used to assign configuration of a new methylphosphonate linkage: preparation of the same diastereomer through multiple synthetic pathways, base hydrolysis, and acid hydrolysis. Hydrolysis of the diastereomerically pure oligomers into component dimers and monomers was followed by chromatographic comparison with control dimers of known configuration. In all cases studied, oligomers with R configurations displayed faster elution from silica gel than did oligomers with the respective S configuration. NMR spectra of individual diastereomers of dACA were studied, revealing characteristic differences in chemical shifts which may prove useful in configurational assignments of longer oligomers. Thus, these data provide a methodological basis for synthesis and configurational assignment of longer methylphosphonate oligomers to use as antisense probes.     

Suppression of food intake by a complement C3a agonist [Trp5]-oryzatensin(5-9)

[Trp5]-oryzatensin(5-9) (WPLPR), an agonist peptide for complement C3a receptor, has been designed based on the C-terminal region of ileum-contracting peptide oryzatensin derived from rice protein. We previously reported that WPLPR has anti-analgesic and anti-amnesic activities after central or oral administration. In this study, we found a novel function of WPLPR on food intake. WPLPR suppressed food intake after intracerebroventricular or intraperitoneal (i.p.) administration at a dose of 3-30 nmol/mouse or 30-300 mg/kg, respectively, in fasted mice. Orally administered WPLPR at a dose of 300 mg/kg also decreased food intake. WPLPR decreased gastric emptying after i.p. injection at a dose of 300 mg/kg. The anorexigenic activity of WPLPR was blocked by cyclooxygenase inhibitor or antagonist for prostaglandin (PG) E receptor EP4 subtype. These results suggest that WPLPR decreases food intake through PGE2 production followed by EP4 receptor activation.     

Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc

The phosphorodiamidate Morpholino oligomers (PMO) are a new class of antisense agents that inhibit gene expression by binding to RNA and sterically blocking processing or translation. In a search for a Morpholino agent that would inhibit cell proliferation, it was found that oligomers directed against c-myc, a gene involved in control of the cell cycle, were effective. The sequence specificity and mechanism of action of one agent were determined. The 20-mer 126 lowers c-myc protein levels in treated cells and arrests cells in G0/G1 of the cell cycle. It also acts at the RNA level to inhibit normal pre-mRNA splicing and instead produces an aberrantly spliced mRNA. Irrelevant and mispair control oligomers indicated that the observed antiproliferative effect was sequence specific. This was confirmed in a reporter gene model system using a c-myc 5'-untranslated region (5'-UTR) fused to a cDNA copy of the insect luciferase gene. We conclude that 126 is acting through an antisense mechanism involving Watson-Crick hydrogen bonding to its target RNA. A specific antisense agent directed against a cell cycle-associated gene mRNA may be useful as a therapeutic in diseases characterized by excess cell proliferation, such as restenosis following balloon angioplasty or cancer.     

Control of ribonucleic acid function by oligonucleoside methylphosphonates

Oligodeoxyribonucleoside methylphosphonates contain nonionic 3'-5' linked methylphosphonate internucleotide bonds in place of the normal charged phosphodiester linkage of natural nucleic acids. These oligomers are resistant to nuclease hydrolysis, can pass through the membranes of mammalian cells in culture and can form stable hydrogen-bonded complexes with complementary nucleotide sequences of cellular RNAs such as mRNA. The oligomers are readily synthesized on insoluble polymer supports. Their chainlength and nucleotide sequence can be determined by chemical sequencing procedures. Oligonucleoside methylphosphonates which are complementary to the 5'-end, initiation codon region, or coding region of rabbit globin mRNA inhibit translation of the mRNA in rabbit reticulocyte lysates and globin synthesis in rabbit reticulocytes. This inhibition is due to the interaction of the oligomers with mRNA and the extent of inhibition is influenced by the secondary structure of the mRNA and the location of oligomer binding site on the mRNA. Oligomers complementary to the initiation codon regions of N, NS and G protein mRNAs of Vesicular stomatitis virus (VSV) inhibit virus protein synthesis in VSV-infected Mouse L-cells. These oligomers do not affect L-cell protein synthesis or growth. Virus protein synthesis and growth can also be selectively inhibited by oligonucleoside methylphosphonates which are complementary to the donor or acceptor splice junctions of virus pre mRNA. An oligomer complementary to the donor splice junction of SV40 large T antigen mRNA inhibits T-antigen synthesis in SV40-infected African green monkey kidney cells but does not inhibit overall cellular protein synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of trichloroethylene on DNA methylation and expression of early-intermediate protooncogenes in the liver of B6C3F1 mice.

Trichloroethylene (TCE) is a multimedia environmental pollution that is carcinogenic in mouse liver. The ability of TCE to modulate DNA methylation and the expression of immediate-early protooncogenes was evaluated. Female B6C3F1 mice were administered 1000 mg/kg TCE by gavage 5 days/week and killed after 5, 12, or 33 days of exposure. Methylation of DNA as 5-methylcytosine was decreased by 5 days of treatment with TCE and remained reduced for 33 days. TCE also decreased the methylation of the promoter regions for the protooncogenes, c-jun and c-myc. The expression of the mRNA for the two protooncogenes was increased between 60 and 120 minutes after administering the last dose of TCE and returned to control level by 24 hours. The expression of the mRNA for c-fos remained undetectable after administering TCE. Hence, TCE decreased the methylation both of total DNA and the promoters for the c-jun and c-myc genes and increased the expression of their mRNA. The decreased methylation and increased expression of the two immediate-early protooncogenes might be associated with TCE-induced increase in cell proliferation and promotion of tumors.     

Effects of Psoralen-Derivatized Oligonucleoside Methylphosphonates on Vesicular Stomatitis Virus (VSV) Protein Synthesis In Vitro and in VSV-Infected Cells

Abstract

Oligodeoxyribonucleoside methylphosphonates (16-mers) targeted to VSV mRNAs were derivatized with 4′-{[N-(aminoethyl)amino]methyl}-4,5′8-trimethylpsoralen. These oligomers specifically inhibit translation of their targeted mRNAs in vitro following UV irradiation of the oligomer-mRNA complexes. Psoralen-derivatized oligonucleoside methylphosphonates are stable in cells and can inhibit VSV protein synthesis in culture following UV-irradiation of VSV-infected cells.

     

Isoflurane Exposure during Mid-Adulthood Attenuates Age-Related Spatial Memory Impairment in APP/PS1 Transgenic Mice

Many in vitro findings suggest that isoflurane exposure might accelerate the process of Alzheimer Disease (AD); however, no behavioral evidence exists to support this theory. In the present study, we hypothesized that exposure of APP/PS1 transgenic mice to isoflurane during mid-adulthood, which is the pre-symptomatic phase of amyloid beta (Abeta) deposition, would alter the progression of AD. Seven-month-old Tg(APPswe,PSEN1dE9)85Dbo/J transgenic mice and their wild-type littermates were exposed to 1.1% isoflurane for 2 hours per day for 5 days. Learning and memory ability was tested 48 hours and 5 months following isoflurane exposure using the Morris Water Maze and Y maze, respectively. Abeta deposition and oligomers in the hippocampus were measured by immunohistochemistry or Elisa 5 months following isoflurane exposure. We found that the performance of both the transgenic and wild-type mice in the Morris Water Maze significantly improved 48 hours following isoflurane exposure. The transgenic mice made significantly fewer discrimination errors in the Y maze following isoflurane exposure, and no differences were found between wild-type littermates 5 months following isoflurane exposure. For the transgenic mice, the Abeta plaque and oligomers in the hippocampus was significantly decreased in the 5 months following isoflurane exposure. In summary, repeated isoflurane exposure during the pre-symptomatic phase not only improved spatial memory in both the APP/PS1 transgenic and wild-type mice shortly after the exposure but also prevented age-related decline in learning and memory and attenuated the Abeta plaque and oligomers in the hippocampus of transgenic mice.     

维生素E和微量元素Se对人肝癌细胞生长、分化及其癌基因表达的影响

本工作观察了体外环境中不同水平的维生素Ｅ和微量元素Ｓｅ对人肝癌细胞株（ＳＭＭＣ－７７２１）生长、分化和其癌基因（Ｎ－ｒａｓ、ｃ－ｍｙｃ）表达水平的影响。实验结果表明：高水平维生素Ｅ（２．４、９．２、２４．０ｎｍｏｌ／Ｌ）和Ｓｅ（０．１５、０．３０、０．６０ｎｍｏｌ／Ｌ）对肝癌细胞的集落形成率具有明显的抑制作用;生化分析显示高水平维生素Ｅ和微量元素Ｓｅ均可明显抑制环境中脂质过氧化的水平,Ｓｅ对癌细胞甲胎蛋白的分泌有明显的抑制作用,而维生素Ｅ作用不明显。细胞原位杂交发现维生素Ｅ浓度为２．４和９．２ｎｍｏ１／Ｌ时对细胞癌基因Ｎ－ｒａｓ的表达具有明显抑制作用;Ｓｅ浓度为０．１５和０．３０ｎｍｏｌ／Ｌ时对癌基因ｃ－ｍｙｃ的表达明显抑制。实验还观察了维生素Ｅ和Ｓｅ之间的叠加效应,结果显示除对环境中脂质过氧化的抑制作用具有叠加效果外,对其他指标没有明显作用。     

Naked DNA prevents soman intoxication

Paraoxonase (Q isoenzyme, PON1) can effectively hydrolyze chlorpyrifos-oxon (CPO), soman, sarin, and other organophosphates. Previous studies had indicated that the levels of serum PON1 in gene therapy with adenoviral vector could decrease the toxicity of CPO. In our study, plasmid pcDNA/PON1 injected into the tail vein of mice gave excellent expression at 24 h after delivery, and PON1 activity decreased gradually along with days. The PON1 activities of mice treated with different doses of the plasmid (150, 300, and 600 μg/mouse) indicated a very good dose-effect relationship. Toxicity study has been performed using one lethal dose of soman (200 μg/kg). The mean death latency of mice pre-treated with 150, 300, 600, and 1200 μg pcDNA/PON1 extended and the mortality decreased vs control mice received the null pcDNA. These results demonstrate that increasing serum PON1 by naked DNA can offer protection toward the acute toxicity of soman.     

Preparation of oligodeoxyribonucleoside methylphosphonates on a polystyrene support.

An efficient procedure is described for synthesizing deoxyribonucleoside methylphosphonates on polystyrene polymer supports which involves condensing 5'-dimethoxytrityldeoxynucleoside 3'-methylphosphonates. The oligomers are removed from the support and the base protecting groups hydrolyzed by treatment with ethylenediamine in ethanol, which avoids hydrolysis of the methylphosphonate linkages. Two types of oligomers were synthesized: those containing only methylphosphonate linkages, d-Np(Np)nN, and those which terminate with a 5' nucleotide residue, dNp (Np)nN. The latter oligomers can be phosphorylated by polynucleotide kinase, and are separated by polyacrylamide gel electrophoresis according to their chain length. Piperdine randomly cleaves the oligomer methylphosphonate linkages and generates a series of shorter oligomers whose number corresponds to the length of the original oligomer. Apurinic sites introduced by acid treatment spontaneously hydrolyze to give oligomers which terminate with free 3' and 5' OH groups. These reactions may be used to characterize the oligomers.     

白细胞介素22对T细胞介导的小鼠肝损伤的治疗作用及初步机制探讨

目的研究人白介素22（IL-22）对T细胞介导的肝损伤小鼠的治疗作用。方法利用刀豆蛋白A（ConA）建立T细胞介导的肝损伤小鼠模型,检测静脉注射IL-22对肝损伤小鼠血清丙氨酸转氨酶（ALT）、天门冬氨酸转氨酶（AST）活性的影响,同时取小鼠肝组织进行病理学检查。并采用半定量RT-PCR方法检测IL-22刺激HepG2和LO2细胞后,对c-myc及Bcl-2基因转录表达水平的影响。结果IL-22明显降低ConA致小鼠急性肝损伤血清ALT、AST值的升高,减轻ConA对肝组织的病理损伤。体外检测IL-22对HepG2和LO2细胞表达c-myc及Bcl-2基因转录水平有促进作用。结论IL-22对T细胞介导的肝损伤小鼠模型具有治疗作用,该作用可能是通过IL-22促进肝细胞的抗凋亡因子的表达实现的。     

Solid-phase synthesis of oligo-2-pyrimidinone-2'-deoxyribonucleotides and oligo-2-pyrimidinone-2'-deoxyriboside methylphosphonates.

A synthetic method was developed for the synthesis of oligodeoxyribonucleotides and oligodeoxyribonucleoside methylphosphonates comprised exclusively of the fluorescent 2-pyrimidinone base for the first time. The method utilized the solid-phase 2-cyanoethylphosphoramidite and methylphosphonamidite chemistry for internucleotide couplings and a baselabile oxalyl linkage to anchor the oligomers onto the CPG support. Cleavage of the oligomers from the support was effected by a short treatment of the support with 5% ammonium hydroxide in methanol at room temperature, without any degradation of the base-sensitive 2-pyrimidinone residues or the base-sensitive methylphosphonate backbone.     

An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation.

To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer. This oligomer was stable in culture and entered cells, forming an intracellular duplex. Incubation of cells with the anti-myc oligomer decreased the steady-state levels of c-myc protein by 50 to 80%, whereas a control oligomer did not significantly affect the c-myc protein concentration. Direct inhibition of c-myc expression with the anti-myc oligomer was associated with a decreased cell growth rate and an induction of myeloid differentiation. Related antisense oligomers with 2- to 12-base-pair mismatches with c-myc mRNA did not influence HL-60 cells. Thus, the effects of the antisense oligomer exhibited sequence specificity, and furthermore, these effects could be reversed by hybridization competition with another complementary oligomer. Antisense inhibition of a nuclear proto-oncogene apparently bypasses cell surface events in affecting cell proliferation and differentiation.     

Natural amyloid-beta oligomers acutely impair the formation of a contextual fear memory in mice

Memory loss is one of the hallmark symptoms of Alzheimer's disease (AD). It has been proposed that soluble amyloid-beta (Abeta) oligomers acutely impair neuronal function and thereby memory. We here report that natural Abeta oligomers acutely impair contextual fear memory in mice. A natural Abeta oligomer solution containing Abeta monomers, dimers, trimers, and tetramers was derived from the conditioned medium of 7PA2 cells, a cell line that expresses human amyloid precursor protein containing the Val717Phe familial AD mutation. As a control we used 7PA2 conditioned medium from which Abeta oligomers were removed through immunodepletion. Separate groups of mice were injected with Abeta and control solutions through a cannula into the lateral brain ventricle, and subjected to fear conditioning using two tone-shock pairings. One day after fear conditioning, mice were tested for contextual fear memory and tone fear memory in separate retrieval trials. Three experiments were performed. For experiment 1, mice were injected three times: 1 hour before and 3 hours after fear conditioning, and 1 hour before context retrieval. For experiments 2 and 3, mice were injected a single time at 1 hour and 2 hours before fear conditioning respectively. In all three experiments there was no effect on tone fear memory. Injection of Abeta 1 hour before fear conditioning, but not 2 hours before fear conditioning, impaired the formation of a contextual fear memory. In future studies, the acute effect of natural Abeta oligomers on contextual fear memory can be used to identify potential mechanisms and treatments of AD associated memory loss.     

Photochemical cross-linking of psoralen-derivatized oligonucleoside methylphosphonates to rabbit globin messenger RNA

Antisense oligodeoxyribonucleoside methylphosphonates targeted against various regions of mRNA or precursor mRNA are selective inhibitors of mRNA expression both in cell-free systems and in cells in culture. The efficiency with which methylphosphonate oligomers interact with mRNA, and thus inhibit translation, can be considerably increased by introducing photoactivatable psoralen derivatives capable of cross-linking with the mRNA. Oligonucleoside methylphosphonates complementary to coding regions of rabbit alpha- or beta-globin mRNA were derivatized with 4'-(aminoalkyl)-4,5',8-trimethylpsoralens by attaching the psoralen group to the 5' end of the oligomer via a nuclease-resistant phosphoramidate linkage. The distance between the psoralen group and the 5' end of the oligomer can be adjusted by changing the number of methylene groups in the aminoalkyl linker arm. The psoralen-derivatized oligomers specifically cross-link to their complementary sequences on the targeted mRNA. For example, an oligomer complementary to nucleotides 56-67 of alpha-globin mRNA specifically cross-linked to alpha-globin mRNA upon irradiation of a solution of the oligomer and rabbit globin mRNA at 4 degrees C. Oligomers derivatized with 4'-[[N-(2-amino-ethyl)amino]methyl]-4,5',8-trimethylpsoralen gave the highest extent of cross-linking to mRNA. The extent of cross-linking was also determined by the chain length of the oligomer and the structure of the oligomer binding site. Oligomers complementary to regions of mRNA that are sensitive to hydrolysis by single-strand-specific nucleases cross-linked to an approximately 10-30-fold greater extent than oligomers complementary to regions that are insensitive to nuclease hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)